Development and validation of an ultra-performance liquid chromatography method coupled with tandem mass spectrometry for determination of alizapride in human plasma

Abstract

The present study describes a novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the estimation of alizapride in human plasma by electro spray ionization in the positive mode using triple quadrupole mass spectrometry using midodrine as an internal standard (IS). Sample pretreatment was carried out with solid-phase extraction using Bond Elut C18 cartridges, resulting in an average recovery of 86.45 ± 0.62 of the investigated compound. The chromatographic separation was performed on an Acquity UPLC BEH shield reversed phase C18 column, using a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.2 mL min⁻¹. The molecular ion of the analyte was detected in positive ionization by multiple reaction monitoring (MRM). The mass transitions of m/z 316.24 → 124.19 and m/z 255.16 → 180.19 were used for detection of alizapride and its internal standard, respectively. The assay exhibited linear ranges from 1 to 1000 ng mL⁻¹ for the analyte in human plasma. The LC-MS/MS method was fully validated for all the other parameters such as selectivity, linearity and range, LLOQ, precision and accuracy, matrix effect, recovery and stability. The lower limit of quantification (LLOQ) of the developed assay method for alizapride was 1 ng mL⁻¹. The intraday and interday variation of the current assay was evaluated with CV% within 4.8%, whereas the mean accuracy ranged from 93.59–100.19%. The samples were stable under the storage conditions for at least a month. In conclusion, the findings of the present study revealed the selectivity and sensitivity of this method for the estimation of alizapride in human samples. The proposed method was successfully applied to determine alizapride in human plasma samples after intramuscular administration of the drug.

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1. Introduction

Alizapride (ALZ) (±)-6-methoxy-N-{[1-(prop-2-en-1-yl)-pyrrolidin-2-yl]methyl}-1H-benzotriazole-5-carboxamide is a dopamine antagonist with prokinetic and antiemetic effects administered as a racemic mixture in the treatment of cancer chemotherapy-induced nausea and vomiting, including postoperative nausea and vomiting.¹ Few HPLC methods were reported for the determination of ALZ. Coulais et al.² developed an HPLC method for the determination of ALZ in human plasma using fluorescence detection. Canal et al.³ and E. Rey et al.⁴ used Coulais method to study the pharmacokinetics of ALZ in cancer patients. A simple HPLC method with UV detection at 230 nm was developed for the determination of ALZ and metoclopramide in human plasma and in urine using solid phase extraction for the sample preparation.⁵ ALZ was also determined with HPLC coupled with fluorescence detection and the method was applied to a dose dependent pharmacokinetic study⁶ in addition Houin et al.⁷ used the same method to determine the absolute intramuscular, oral, and rectal bioavailability of ALZ. Most of the reported methods for determination of ALZ use liquid–liquid extraction^2–4,6,7 or lack IS.^6,7 Tamaro et al.⁸ reported an HPLC method with UV detection for the determination of ALZ in drug powder and drug product. Sample preparation method is a key step in bioanalytical method development that leads to improved selectivity and sensitivity. Solid phase extraction (SPE) produces the cleanest samples (compared to liquid–liquid extraction (LLE) or protein precipitation (PP)) and has the advantages of the avoidance of emulsion formation, low volume of solvents and has a high through-put performance (compared to LLE).⁹ UPLC technology offers significant advantages in resolution, speed and sensitivity for analytical applications, especially when coupled with mass spectrometers. Its high chromatographic resolution enables narrower chromatographic peak shapes and better chromatographic separation, thereby providing improved sensitivity, shorter analysis times, and reduced matrix effects (due to the better separation of drug-related components from endogenous components). Therefore, UPLC-MS is suitable for forensic and clinical analyses which require high sensitivity and analytical speed.⁹ Aiming to develop a bioanalytical method capable of determining ALZ plasma concentration from the time of administration till elimination; a sensitive and selective validated UPLC-MS/MS method, using fast SPE cartridges with small amounts of sample plasma volume and organic solvents, was developed.

2. Experimental

2.1. Equipment

Acquity waters UPLC system equipped with a quaternary pump, autosampler and a tandem mass TQ detector (USA) was used. Separation was performed on Acquity UPLC BEH shield RP-C18 column (150 × 2.1 mm, 1.7 μm particle size).

2.2. Materials and reagents

2.2.1. Pure standards. Working standard of ALZ was obtained as a kind gift from Minapharm Company-Egypt; its purity was certified to be 99.8%.

Working standard of midodrine HCl (internal standard) was obtained as a kind gift from Nile Company-Egypt; its purity was certified to be 99.8%.

2.2.2. Pharmaceutical formulation. Nausilex® ampoules claimed to contain 55.8 mg of alizapride hydrochloride equivalent to 50 mg of ALZ base (Minapharm, Egypt) were purchased from the local market.

2.2.3. Chemicals and reagents. All solvents used were HPLC grade. Acetonitrile, methanol, and formic acid eluent additive for LC/MS were purchased from Sigma Aldrich, Egypt. Ultra-pure water (18 Mohm cm) was obtained from an Elga Ultrapure Q apparatus.

Hydrochloric acid, sodium hydroxide, and potassium di-hydrogen phosphate were purchased from El-Nasr Company, Egypt; phosphate buffer solutions of pH 4.5 and 8.5 were prepared as mentioned in the British Pharmacopeia.¹⁰

Human Plasma was obtained from El-Kasr El-Aini blood bank, Egypt. Solid phase extraction (SPE) cartridges (Bond Elut C18, 100 mg 2 mL) were purchased from Agilent (USA).

2.3. Stock and working solutions

Two separate working solutions were used for the preparation of calibration curve standards and quality control samples. Stock solution of ALZ was prepared in water (1 mg mL⁻¹). This solution was further diluted with water to obtain two different concentrations: 1 and 100 μg mL⁻¹ of ALZ. The two solutions were appropriately diluted with water so as to obtain working solutions for calibration standards as; 10, 100, 500, 1000, 2500, 5000, 7500, 10 000 ng mL⁻¹; and working solutions for quality control samples as: 30 ng mL⁻¹ (QCL, low quality control), 4000 ng mL⁻¹ (QCM, medium quality control) and 8000 ng mL⁻¹ (QCH, high quality control).

Stock solution of the internal standard was prepared by dissolving 10 mg of midodrine HCl in 10 mL water. This solution was further diluted with water to obtain a working solution of 100 μg mL⁻¹ of internal standard. All solutions were stored at 4–8 °C.

2.4. Preparation of calibration standards and quality control samples

To 450 μL of the drug free human plasma, 50 μL of working solutions of ALZ and 75 μL of internal standard were added to yield final respective concentrations of 1, 10, 50, 100, 250, 500, 750 and 1000 ng mL⁻¹ of ALZ in human plasma. QC samples (3, 400, 800 ng mL⁻¹) were prepared in a similar manner. All samples were vortexed for 1 minute before extraction.

2.5. Extraction protocol

After spiking the plasma with the drug and internal standard, 1.5 mL buffer pH 8.5 was added and the solutions were vortexed for 1 minute. The SPE cartridges were conditioned by rinsing with 2 mL methanol then 2 mL buffer pH 8.5 then the samples were loaded. The cartridges were then washed with buffer pH 4.5 to elute the plasma proteins and other impurities leaving the drug and internal standard on the cartridge. ALZ and the internal standard were finally eluted with 2 mL acidified methanol pH 3. The collected solutions were evaporated to dryness and then vortexed after reconstitution with 150 μL water before injection into the LC-MS/MS system.

2.6. Chromatographic and mass spectrometry

A linear gradient elution was carried out using a mobile phase consisting of acetonitrile (A) and water containing 0.1% formic acid (B) with flow rate of 0.2 mL min⁻¹. The gradient began with 15% eluent A and 85% eluent B for 1 min and was changed to 10% eluent B and 90% eluent (A) and remained at this ratio for 2 min. The temperatures of analytical column and the autosampler were both set at room temperature.

Typical mass spectrometric conditions were: capillary voltage, 3500 V; ion source temperature, 120 °C; desolvation temperature, 400 °C; cone voltage, 30 V for ALZ and 5 for midodrine; collision energy, 30 V (Table 1). Multiple reaction monitoring (MRM) scanning in positive ionization mode was used to monitor the transition of m/z 316.24 → 124.19 for ALZ and 255.16 → 180.19 for midodrine (Fig. 1).

Table 1 Mass conditions for alizapride and midodrine

Compound	Precursor ion (m/z)	Product ion (m/z)	Cone voltage	Collision energy	Dwell (s)	Capillary voltage	Ion source temperature	Desolvation temperature
Alizapeide	316.24	124.19	30 V	30 V	0.146	3500 V	120 °C	400 °C
Midodrine	255.16	180.19	5 V

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Fig. 1 Mass spectrum of (A) alizapride and its fragmentation product (B) midodrine (IS) and its fragmentation product.

2.7. Bio-analytical method validation

Method validation was performed according to the FDA Guidance for Industry Bioanalytical Method Validation.¹¹

2.7.1. Selectivity. Plasma samples (six blanks) were chromatographed prior to use to check for endogenous components, which might interfere with ALZ or IS in the lots of human plasma used for the preparation of calibration standards and quality control samples.

2.7.2. Linearity and range. For checking linearity, six calibration curves were constructed from eight non-zero samples covering the total range 1–1000 ng mL⁻¹. In addition, a blank sample (a plasma sample processed without the IS), a zero sample (a plasma processed with the IS) were also analyzed to ensure the absence of interferences. These two samples were not used to construct the calibration curve. The calibration curves were generated using the analyte to IS peak area ratios by least-squares linear regression on consecutive days. The acceptance criterion for a calibration curve was a correlation coefficient (r) of 0.99 or better, and that each back-calculated standard concentration must be within 15% deviation from the nominal value except at the LLOQ, for which the maximum acceptable deviation was set at 20%. At least 67% of non-zero standards were required to meet the above criteria, including LLOQ and upper limit of quantification.

2.7.3. Lower limit of quantification (LLOQ). LLOQ was established as the lowest calibration standard with an accuracy of 80–120% and precision less than 20%. The signal to noise ratio of the LLOQ signal should not be less than 10.

2.7.4. Precision and accuracy. Precision and accuracy of the method were assessed by analyzing QC samples at the concentrations mentioned above along with the calibration curve. To evaluate intra-day precision and accuracy, 6 samples of each QC concentration were processed and analyzed on the same day. To evaluate inter-day precision and accuracy, 6 samples of each QC concentration were analyzed per day on different days. Percent accuracy was evaluated as: [(mean found concentration)/(nominal concentration)] × 100. Precision was expressed as the % CV. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV.

2.7.5. Recovery and matrix effect. B. K. Matuszewski et al. discussed the recovery and matrix effect in full detail.¹² Recovery of ALZ from the extraction procedure was determined by comparing the peak area of ALZ (three concentrations, each of low, medium and high QCs) in spiked plasma samples that were exposed to the whole extraction procedure (pre-extraction samples), with the peak area of ALZ in samples spiked at the end of the extraction procedure (post extraction samples).

Blank plasma samples from six sources were extracted and then spiked with the analyte and IS to assess the matrix effect. The corresponding mean peak area of the analyte in spiked plasma post-extraction (A) at low, medium and high QC levels were then compared with those of the corresponding standard samples (B) at equivalent concentrations. This is defined as the matrix effect. A value of 100% is indicative of no matrix effect, a value > 100% suggests ionization enhancement and a value < 100% suggests ionization suppression.¹³

2.7.6. Stability of the analytes. The stability of the analytes in human plasma was investigated in four ways in order to characterize each operation during the process of analysis of samples: short term stability at room temperature, post-preparative stability in the autosampler, freeze–thaw stability and long-term stability at −80 °C. For all stability studies; the quality control samples, QCL, QCM and QCH were used. Three replicates of those quality controls samples were freshly processed and analyzed in a single run to serve as time zero samples (comparison or fresh samples).
Short term storage stability. Three replicates of each QC samples were left at room temperature (stability samples). All samples remained on the bench top for a time exceeding the maximum period of time expected for routine sample preparation (6 h). After 6 hours, a calibration curve was freshly processed and analyzed with all stability samples. The concentration of the drug in the stability samples were compared to the fresh ones at equivalent concentration.
Post preparative stability. Three replicates of each QC sample were prepared, immediately processed and stored in the thermostated autosampler (25 °C). The samples were injected after 20 h, the expected longest storage times of the samples in autosampler before injection. A calibration curve was freshly processed and analyzed with the stability samples in a single run. The concentration of the drug in the stability samples was compared to the fresh ones at equivalent concentration.
Freeze and thaw stability. Stability of ALZ was assessed in plasma samples subjected to three freeze–thaw cycles of −80 °C during 24 h. Three replicates of each QC sample were prepared and subjected to three cycles of freeze–thaw operations in three consecutive days. After the third cycle the samples were analyzed against calibration curve of the day. The mean concentration calculated for the samples subjected to the cycles and the fresh ones at equivalent concentration were compared.
Long-term stability. Three replicates of each QC sample were subjected to freeze storage (−80 °C) for 30 days. Storage stability was defined, comparing sample concentration to the fresh ones at equivalent concentration. The values were calculated against calibration curve of the day and the mean values for the stored samples and nominal concentrations were compared.

The requirement for stable analytes was that the difference between mean concentrations of the tested stability samples in various conditions and nominal concentrations (fresh samples) had to be in ±15% range.

% Change = (stored − fresh)/fresh × 100

3. Results and discussion

The aim of this work was to develop a sensitive LC/MS/MS method for determination of ALZ in human plasma that is suitable for bio-equivalence, bioavailability and therapeutic drug monitoring using SPE for sample clean up.

3.1. Chromatographic and mass spectrometric conditions

To optimize the proposed LC/MS/MS method, the effect of several chromatographic parameters was studied in order to achieve the best separation and retention for the target analytes. These included the type of organic modifier, pH of aqueous solution and organic modifier – aqueous ratio. These parameters were optimized based on the peak shape, peak intensity/area and retention time for ALZ and Mido. Several experiments were performed using different mobile phases consisting of methanol or acetonitrile as the organic phase and water with different concentrations of formic acid and acetic acid (0.1–0.5%). The mobile phase containing 0.1% formic acid gave higher detection sensitivity for both ALZ and the IS. Several isocratic mobile phase ratios were tested but all give poor peak shape and symmetry so gradient elution was tried. Finally, the best chromatographic separation was achieved using a gradient mobile phase system composed of acetonitrile (A) and water containing 0.1% formic acid (B) with the flow rate of 0.2 mL min⁻¹. The gradient began with 15% eluent A and 85% eluent B for 1 minute and was changed to 10% eluent B and 90% eluent (A) and remained at this ratio for 2 minutes. Other parameters such as column temperature and flow rate were studied in order to get a fast and reliable separation. The best results were observed at room temperature and 0.2 mL min⁻¹ as the flow rate. Under these conditions the Rt of ALZ and IS were 2.49 and 2.45, respectively.

The full Q1 scan of ALZ and midodrine was acquired in positive ion mode by infusing the standard solutions into electro spray ionization source. The product ion [M + H]⁺ mass spectra of these two compounds are shown in Fig. 1. The most sensitive mass transitions were from m/z 316.24 → 124.19 for ALZ and m/z 255.16 → 180.19 for the IS. LC-MRM mode provides sensitivity and selectivity requirements for analytical methods used for the detection of plasma drug concentrations. Thus, the MRM technique was chosen for the method development. The MRM parameters were optimized to maximize the response of each of precursor/product transition. The parameters are presented in Table 1.

3.2. Extraction procedure

The availability of a wide range of cartridges and solvents make SPE suitable for many analytes with various properties. A reversed phase C18 SPE cartridges was found optimum for ALZ extraction. To optimize the extraction parameters, it was found that maintaining the sample pH at 8.5 was suitable for both ALZ and IS retention on the cartridge as at this pH ALZ is present in its non-ionized form (pK_a = 7.5). Buffer pH 4.5 was found to be suitable for the washing process to decrease the retention of plasma proteins and other impurities where at this pH, ALZ was still retained on the cartridges. The best elution solvent for ALZ and the IS was found to be acidified methanol pH 3 as ALZ will be totally ionized and cannot bind further to C18 cartridges.

Choosing a suitable IS is an important aspect to achieve acceptable method performance, especially with LC/MS/MS, where matrix effects can lead to poor analytical results due to ionization suppression or enhancement. Ideally, isotopically labeled internal standards for the analyte should be used, but these are not always commercially available and expensive. Amisulpride which is structurally related to ALZ was initially tried but it failed to be retained on the cartridge at the same pH of ALZ as it has a different pK_a of 9.37. While midodrine (pK_a of 7.8) was successfully recovered by the studied extraction procedure thus chosen to be the internal standard.

3.3. Method validation

3.3.1. Selectivity. Fig. 2 shows the typical chromatograms of a blank, a spiked plasma sample with ALZ and IS. No interference in the blank plasma samples was observed from endogenous substances in drug-free human plasma at the retention time of the analyte. Similarly, no interference from the IS to the MRM channel of the analyte was observed.

Fig. 2 Representative (A) blank plasma samples, (B) spiked plasma sample at LLOQ (1 ng mL⁻¹) with the IS, (C) spiked plasma sample at QCH (800 ng mL⁻¹) with the IS and (D) real plasma sample at 10 minutes spiked with the IS.

3.3.2. Linearity and LLOQ. The linear regression of the peak area ratios versus concentrations was fitted over the concentration range of 1–1000 ng mL⁻¹ for ALZ. The correlation coefficient (r) exceeded 0.99, showing a good linearity among the concentration range.

The lower limit of quantification (LLOQ) was 1 ng mL⁻¹ for ALZ where the mean response for ALZ peak at this concentration was 10-fold greater than the mean response for the peak in the blank human plasma samples at the retention time of the analyte. The % RSD was 15.49 at LLOQ level, which was within the accepted limits.

3.3.3. Recovery and matrix effect. The suggested method yielded a recovery of 87.13 ± 6.18% in plasma samples spiked with 800 ng mL⁻¹ ALZ and 81.70 ± 2.95% for the IS. Recoveries of the analytes and IS were satisfied, consistent, precise and reproducible (Table 2). Therefore, the method has proved to be robust in high-throughput bioanalysis. In this study, the presence of plasma suppressed the ion signal and these results were consistent in all the six human plasma batches (Table 2).

Table 2 Recovery of alizapride and midodrine in plasma

	Conc. (ng mL^−1)	% Recovery (n = 3)	% Matrix effect (n = 3)
Alizapride	3	86.28 ± 6.31	85.23 ± 3.61
	400	85.93 ± 8.49	85.06 ± 3.89
	800	87.13 ± 6.18	88.23 ± 2.303
Midodrine	750	81.70 ± 2.59	71.27 ± 6.70

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3.3.4. Precision and accuracy. Table 3 summarizes the intra- and inter-day precision and accuracy for the analyte in QC samples. The intra- and inter-day RSD were below 4.8%. All the values were within the accepted range and the method was precise. Regarding accuracy, all the values were in the accepted range of ± 15%.

Table 3 Precision and accuracy for determination of ALZ in human plasma

Nominal conc. (ng mL^−1)	Intra-day (n = 6)		Inter-day (n = 9)
	Accuracy%	RSD%	Accuracy%	RSD%
3	100.19	3.9	98.82	3.81
400	96.41	4.8	96.50	4.61
800	93.59	4	94.69	4.41

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3.3.5. Assessment of stability. ALZ was found to be stable in plasma at room temperature for 6 hours which is the maximum time for sample preparation. No significant degradation of ALZ and IS was observed when the extracted samples were kept in the autosampler for 12 hours. Upon three freeze/thaw cycles, the maximum% change was 2.03. ALZ was also found to be stable under the long term storage conditions (−80 °C) for at least 30 days which is long enough to cover the duration of long studies. (Table 4)

Table 4 Stability of alizapride under different conditions

Stability	Concentration added (ng mL^−1)	Mean concentration found (ng mL^−1)	% CV	% Accuracy
Short term (8 hours)	3	2.99	2.74	99.89
	400	380.23	4.84	95.06
	800	751.45	6.83	94.93
Freeze–thaw (three cycles)	3	2.88	4.55	95.93
	400	384.52	4.58	96.13
	800	737.35	2.14	92.17
Auto sampler (30 hours)	3	2.96	4.40	98.73
	400	375.94	2.64	93.99
	800	748.52	5.11	93.57
Long term (30 days)	3	2.97	3.83	98.89
	400	384.79	5.81	96.20
	800	732.21	5.06	91.53

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3.4. Comparison with previously published methods

This work describes an UPLC method which has many advantages over previously published HPLC methods as this technique increases separation efficiency due to lower particle size, is more echo-friendly as the flow rate is 0.2 mL min⁻¹ and therefore consumes less solvents. Furthermore, the proposed method is more sensitive than all previously published methods; LLOQ 1 ng mL⁻¹. SPE is much more advantageous than LLE procedures described in ref. 2–4, 6 and 7 as it gives the cleanest samples, lower solvent consumption and avoidance of emulsion formation. Furthermore, the SPE procedure described is much simpler than that reported by Jong et al.⁵

3.5. Application to pharmacokinetic study

The method was applied to determine the plasma concentration of ALZ in real plasma samples obtained at different time intervals following a single 50 mg intramuscular administration (IM) to a 40 years old male adult. The blood samples were collected into the heparinized tubes at 0, 0.083, 0.16, 0.25, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8 and 24 hours post dose. Five milliliters of venous blood were withdrawn. Blood samples were transferred immediately to heparinized tubes, centrifuged for 10 minutes at 3200 rpm (at 4 °C) and the separated plasma transferred to Eppendorf tubes and stored at −80 °C until the day of analysis. Plasma samples were processed as described above to determine the concentration of ALZ. Fig. 3 shows the plasma concentration–time profile after ALZ IM administration. Maximum concentration was 954.45 ng mL⁻¹ and occurred within 20 minutes after injection. Area under curve (AUC_0–24) was found to be 6133.47 ng mL⁻¹ and t_1/2 was found to be 4.19 hours.

Fig. 3 Plasma concentration–time curve following an intra-muscular dose of 50 mg of alizapride ampoules (Nausilex®).

The study was approved by faculty of Pharmacy, Cairo University ethics committee.

4. Conclusions

The present study developed and validated a facile, specific, and sensitive UPLC-MS/MS method for the determination of ALZ in human plasma. The LLOQ was 1 ng mL⁻¹, which was sensitive enough to determine the low concentration of ALZ in human plasma. SPE pretreatment was very convenient and provided high recovery (about 85%). This method has been successfully applied to determine the plasma concentration of ALZ following IM administration of 50 mg drug.

Acknowledgements

This work was funded by Faculty of Pharmacy-Cairo University.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra10386j

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